Primary cilia organize signaling pathways such as vision, olfaction and Hedgehog signaling. The movements of signaling receptors into, inside and out of cilium are critical for the correct regulation of these pathways, yet our understanding of the basic mechanisms governing signaling receptor trafficking through cilia remains fragmentary. Past work from the lab identified and characterized the BBSome, a protein complex that ferries signaling receptors out of cilia. The relevance of the BBSome to human health and disease is evidence by the fact that BBSome dysfunction causes Bardet-Biedl Syndrome (BBS), a hereditary disease characterized by obesity, retinal degeneration, polydactyly and kidney malformations. The major goal of this proposal is to determine how the BBSome selects signaling receptors for removal from cilia. The emphasis in this funding period will be on investigating the role of ubiquitin in tagging membrane proteins for removal from cilia. Preliminary data indicate the existence of a ciliary machinery that recognizes activated GPCRs, ubiquitinates them and sorts ubiquitinated GPCRs out of cilia. We will characterize the molecules acting at each of these steps using quantitative assays for signal-dependent GPCR exit. The hierarchical ordering of the molecular cogs and levers that affix and read ubiquitin on proteins that need to exit cilia promises to uncover a multi-step pathway reminiscent of the ESCRT machinery responsible for degradative sorting. Finally, the fate of GPCRs that exit cilia will be tracked by single-molecule imaging to determine whether endocytosis is coupled to ciliary exit or whether GPCRs instead diffuse into the plasma membrane after exiting cilia. The proposed studies will cast new light on how the ciliary abundance of proteins is regulated and open the door to a mechanistic investigation of ciliary quality control.